Size regulation of double and half embryos in the mice musculus

Genetic chimeras made by aggregating early mouse embryos have many uses in developmental biology and have also provided insights into embryonic growth regulation. There is an indication that the embryo can regulate for an increase in size because although aggregation chimeras are twice as big as normal embryos when made, they are born of normal size. Upward regula..... tion of size reduced embryos is also possible. Half embryos made by the isolation or destruction of one of the blastomeres of a 2-cell embryo are also born of normal size. Little is known about the timing or the mechanism of this size regulation. In this study, the timing of size regulation in double and half embryos was clearly established by comparison of cell numbers derived from serial reconstruction of light microscope sections of control and experimental embryos. It was shown that size regulation in double embryos occurred around 6dl6h and in half embryos by 7dOh. Size regulation occurred in all tissues at the same time indicating a single control mechanism for the entire embryo. More detailed examination of the growth of double embryos revealed that size regulation occurred by alteration in cell cycle length~ No excessive cell death was found in double embryos compared to the controls and continuous labelling with [3H] thymidine showed no large non-dividing cell population in double embryos. However, a comparison of the mitotic index of double and control embryos after colcemid treatment, revealed a large difference between the two around 5dl6h to 6d16h. During this period, control embryos underwent a proliferative burst not shown by the double embryos. The mechanism for cell cycle control is not clear; it may be intrinsic to the embryo or determined by the uterine environment. Evidence was found suggesting that differentiation in the postimplantation embryo was cell number dependent. The timing of differentiative events was examined in half, double and control embryos. Proamnion formation, which occurs prior to size regulation, occurs at the same cell number but at different times in the three groups of embryos. However mesoderm which appears after size regulation was seen at the same time in all grollps of embryos.

Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

Pyruvate dehydrogenase activity in response to skeletal muscle contraction at two stimulation frequencies in pyruvate dehydrogenase kinase 2 knockout mice

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PD H is deactivated by a set of PD H kinases (PD K 1-4) with PDK2 and 4 being the predominant isoforms in skeletal muscle. PDK2 is highly sensitive to pyruvate inhibition, and is the most abundant isoform, while PDKI and 4 protein content are normally lower. This study examined the PDK isoform content and PDHa activation in muscle at rest and 10 and 40 Hz stimulation from PDK2 knockout (PDK2KO) mice to delineate the role of PDK2 in activating the PDH complex during low and moderate intensity muscle contraction. PDHa activity was lower in PDK2KO mice during contraction while total PDK actitvity was -4 fold lower. PDK4 protein was not different, however PDKI partially compensated for the lack of PDK2 and was -56% higher than WT. PDKI is a very potent inhibitor of the PDH complex due to its phosphorylation site specificity and allosteric regulation. These results suggest that the site specificity and allosteric regulatory properties of the individual PDK isoforms are more important than total PDK activity in determining transformation of the complex and PDHa activity during acute muscle contraction.

Isoflavone exposure throughout suckling results in improved adult bone health in mice

Exposure to isoflavones (ISO), abundant in soy protein infant formula, for the first 5 days of life results in higher bone mineral density (BMD),greater trabecular connectivity and higher fracture load of lumbar vertebrae (LV) at adulthood. The effect of lengthening the duration of exposure to ISO on bone development has not been studied. This study determined if providing ISO for the first 21 days of life, which more closely mimics the duration that infants are fed soy protein formula, results in higher BMD, improved bone structure and greater strength in femurs and LV than a 5-day protocol. Female CD-1 mice were randomized to subcutaneous injections of ISO (7 Q1 mg kg/body weight/day) or corn oil from postnatal day 1 to 21. BMD, structure and strength were measured at the femur and LV at 4 months of age, representing young Q2 adulthood. At the LV, exposure to ISO resulted in higher (P,0.05) BMD, trabecular connectivity and fracture load compared with control (CON). Exposure to ISO also resulted in higher (P,0.05) whole femur BMD, higher (P,0.05) bone volume/total volume and Q3 lower (P,0.05) trabecular separation at the femur neck, as well as greater (P,0.05) fracture load at femur midpoint and femur neck compared with the CON group. Exposure to ISO throughout suckling has favorable effects on LV outcomes, and, unlike previous studies using 5-day exposure to ISO, femur outcomes are also improved. Duration of exposure should be considered when using the CD-1 mouse to model the effect of early life exposure of infants to ISO.

EARLY LIFE EXPOSURE TO GENISTEIN AND DAIDZEIN DISRUPTS STRUCTURAL DEVELOPMENT OF REPRODUCTIVE ORGANS IN FEMALE MICE

In mice, exposure to isoflavones (ISO), abundant in soy infant formula, during the first 5 d of life alters structural and functional development of reproductive organs. Effects of longer exposures are unknown. The study objective was to evaluate whether exposure to a combination of daidzein and genistein in the first 10 compared to 5 d of life results in greater adverse effects on ovarian and uterine structure in adult mice. Thirteen litters of 8–12 pups were cross-fostered and randomized to corn oil or ISO (2 mg daidzein + 5 mg genistein/kg body weight/d) for the first 5 or 10 d of life. The 10-d protocol mimicked the period when infants are fed soy protein formula (SPF) but avoids the time when suckling pups can consume the mother’s diet. Body and organ weights and histology of ovaries and uteri were analyzed. There were no differences in the ovary or uterus weight, number of ovarian follicles, number of multiple oocyte follicles, or percent of ovarian cysts with 5 or 10 d of ISO intervention compared to respective controls. The 10-d ISO group had higher body weights from 6 d to 4 mo. of age and a higher percent of hyperplasia in the oviduct than the respective control. Lower numbers of ovarian corpus lutea and a higher incidence of abnormal changes were reported in the uteri of both ISO groups compared to their respective controls. Five- and 10-d exposure to ISO had similar long-lasting adverse effects on the structures of ovaries and uterus in adult mice. Only the 10-d ISO exposure resulted in greater body weight gain at adulthood.